UPLC-PDA factorial design assisted method for simultaneous determination of oseltamivir, dexamethasone, and remdesivir in human plasma

A green and simple UPLC method was developed and optimized, adopting a factorial design for simultaneous determination of oseltamivir phosphate and remdesivir with dexamethasone as a co-administered drug in human plasma and using daclatasvir dihydrochloride as an internal standard within 5 min. The separation was established on UPLC column BEH C18 1.7 μm (2.1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times $$\end{document}× 100.0 mm) connected to UPLC pre-column BEH 1.7 μm (2.1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times $$\end{document}× 5.0 mm) at 50 °C with an injection volume of 10 μL. The photodiode array detector (PDA) was set at three wavelengths of 220, 315, and 245 nm for oseltamivir phosphate, the internal standard, and both dexamethasone and remdesivir, respectively. The mobile phase consisted of methanol and ammonium acetate solution (40 mM) adjusted to pH 4 in a ratio of 61.5:38.5 (v/v) with a flow rate of 0.25 mL min−1. The calibration curves were linear over 500.0–5000.0 ng mL−1 for oseltamivir phosphate, over 10.0–500.0 ng mL−1 and 500.0–5000.0 ng mL−1 for dexamethasone, and over 20.0–500 ng mL−1 and 500.0–5000.0 ng mL−1 for remdesivir. The Gibbs free energy and Van't Hoff plots were used to investigate the effect of column oven temperatures on retention times. Fluoride-EDTA anticoagulant showed inhibition activity on the esterase enzyme in plasma. The proposed method was validated according to the M10 ICH, FDA, and EMA’s bioanalytical guidelines. According to Eco-score, GAPI, and AGREE criteria, the proposed method was considered acceptable green.

*Notes on Table S2: It was suggested to choose favipiravir (FAV) [1], DAC [2], or ledipasivir (LED) [3] as the internal standard (IS).Different conditions were used to develop the method.The studied drugs (OSTP, DEX, and REM) with DAC as IS were tried to be simultaneously separated by a mobile phase consisting of a high amount of methanol percentage (MOH%) with 8 mM ammonium acetate at pH 7. The flow rate was set at 0.05 mL min -1 , and the column oven temperature was 25 o C (Table S2, trial 1).
It was observed that the order of eluted peaks was OSTP, DEX, REM, and then DAC.The run time was very long, and all resulted peaks were very broad.Also, OSTP was very tailed (USP peak tailing, T = 2.2).So, it was recommended to increase the flow rate to reduce the run time (Table S2, trials 2-5).It was observed that the run time and the width of the peaks were reduced.But unfortunately, REM and DAC (IS) peaks overlapped.So, FAV and LED were tried as IS (Table S2, trials 6-18).LED peak was very bad in shape and was overlapped with the solvent peak, while FAV showed promising results.FAV showed good system suitability parameters at separation conditions of MOH with 50 mM ammonium acetate (pH 4) at a ratio of 5:95, flow rate of 0.4 mL min -1 , and column oven temperature of 25 o C (Table S2, trial 18).The purpose of the high strength (50 Mm) with low pH (4) of ammonium acetate was to mask the free silanol groups and hence delay FAV elution (capacity factor, K' = 2.8) as its log P is very small (nearly 0.5) (Table S3), and to reduce T of FAV as ammonium acetate can improve peak shape not only by deactivating the free silanol groups but also by forming ion pairs with the analyte [4].Unfortunately, this separation condition of 5% MOH with 95% ammonium acetate would dramatically delay the elution of OSTP, DEX, and REM as they have high log P (more than 1) (Table S3).So, the idea of developing a gradient method to elute the highly polar FAV (IS) with the less polar compounds (OSTP, DEX, and REM) in a reasonable time (less than 10 min) was recommended.This gradient method would start with a low MOH percentage in the mobile phase to elute FAV (IS) and end with a high MOH percentage to elute the studied drugs.Different trials were used to develop this gradient method (Table S2, trials 19-27), but a highly unstabilized baseline with a very low FAV capacity factor (K' ≈ 0.3) and badly shaped peaks were identified.
So, returning to the isocratic method was recommended.DAC was tried again as IS.It has a good log P (Table S3) to be eluted with the studied drugs under isocratic conditions.But the main problem was its overlapping with REM (Table S2, trials 2-5).So, different trials were used to solve this problem (Table S2, trials 28-35).Deionized water (pH 7) as an aqueous mobile phase with MOH at a ratio of 60:40 was first tried as a mobile phase.At a flow rate of 0.4 mL min -1 and a column oven temperature of 25 o C, the order of the eluted peaks was DEX, OSTP, REM, and then DAC (Table S2, trial 28).DEX had a good capacity factor (K' = 2.9), but the OSTP peak was tailed (T = 1.9) and had a high USP resolution (R = 13.8).DAC (IS) was eluted after the tested drugs, increasing the run time to 7 min.So, it was recommended to try the acidified, deionized water (pH 4) with phosphoric acid (Table S2, trial 29) to increase OSTP and DAC ionization (Table S3) and at the same time mask the free silanol groups in the stationary phase.This would lead to decreasing the interaction of the ionized OSTP and DAC molecules with free silanol groups.And hence, OSTP peak tailing and resolution would be decreased as it would be eluted a little earlier.Also, ionized DAC molecules would be eluted before the unionized REM molecules, leading to a shorter run time.At Trial 29 (Table S2), it was observed that OSTP peak tailing was relatively decreased, but DAC peak tailing was increased.So, it was suggested to use 10 mM ammonium acetate at pH 4 (Table S2, trial 30) to increase the masking effect of the free silanol groups and hence decrease the peak tailings.At Trial 30 (Table S2), it was observed that DAC and OSTP tailings were decreased and that OSTP was eluted before DEX due to lower interactions between the ionized OSTP and free silanol groups.So the final order at Trial 30 (Table S2) was OSTP, DEX, DAC (IS), and then REM with a separation condition of MOH: ammonium acetate (pH 4) =40:60, flow rate 0.4 mL min -1 , and column oven temperature of 25 o C. Different strengths of ammonium acetate with different pHs (3-7) were tried (Table S2, trials 31-35).It was observed that the retention times of OSTP and DAC were greatly affected by pH of the ammonium acetate aqueous mobile phase.So, it was concluded that ammonium acetate (10-50 mM) as an aqueous mobile phase solution adjusted at pH 4 with phosphoric acid showed the most suitable peak order (OSTP, DEX, DAC, and then REM) with good system suitability parameters.Finally, Trials 35-38 (Table S2) were tested in order to identify the factorial design factors and their levels.
Table S3: Physical characters of oseltamivir phosphate, dexamesasone, remdesivir, favipiravir, daclatasvir dihydrochloride, and ledipasivir.a The P-value determines the appropriateness of rejecting the null hypothesis in a hypothesis test.P-values range from 0 to 1.A commonly used -level value is 0.05.If the p-value of a test statistic is less than -level, the null hypothesis is rejected.b In practice, the p-value for the intercept term is unimportant.However, the intercept term in the model would still be kept even if the p-value isn't less than a-level (0.05).c R 2 (R-squared) is the percentage of response variable variation that is explained by its relationship with one or more predictor variables.In general, the higher the R 2 , the better the model fits your data.R 2 is always between 0 and 100%.It is also known as the coefficient of determination or multiple determinations (in multiple regressions).d Adjusted R 2 is the percentage of response variable variation that is explained by its relationship with one or more predictor variables, adjusted for the number of predictors in the model.This adjustment is important because the R 2 for any model will always increase when a new term is added.A model with more terms may appear to have a better fit simply because it has more terms.However, some increases in R 2 may be due to chance alone.The adjusted R 2 is a useful tool for comparing the explanatory power of models with different numbers of predictors.The adjusted R 2 will increase only if the new term improves the model more than would be expected by chance.It will decrease when a predictor improves the model less than expected by chance.
e Predicted R 2 is used in regression analysis to indicate how well the model predicts responses for new observations, whereas R 2 indicates how well the model fits your data.Predicted R 2 can prevent overfitting the model and can be more useful than adjusted R 2 for comparing models because it is calculated using observations not included in model estimation.Overfitting refers to models that appear to explain the relationship between the predictor and response variables for the data set used for model calculation but fail to provide valid predictions for new observations.Predicted R 2 is calculated by systematically removing each observation from the data set, estimating the regression equation, and determining how well the model predicts the removed observation.The predicted R 2 ranges between 0 and 100%.Larger values of predicted R 2 suggest models of greater predictive ability.b Each result is the average of five determinations.
c The acceptance criteria were ± 15 % of the nominal concentration's values for QC samples and with ± 20 % for LLOQ.d The acceptance criteria should not exceed 15 % for QC samples except for LLOQ which should be less than 20 %.
e Each result is the average of nine determinations over three days (n=3 per day).a Each result is the mean of four results prepared from blank plasma from four different sources (plasma A, B, C, and D) in percentage ± their standard deviation in percentage.

Fig. S3 :
Fig. S3:The nine runs of 23 FFD experiments with centerpoint experiment of the analyzed drugs.

Fig. S5 :
Fig. S5: GAPI assessment of the green profile of the proposed method.

Fig. S6 :
Fig. S6: Result of AGREE analysis for the proposed method. Fig.S1

Table S4 :
23FFD experimental factorial designs with centerpoint experiment and their dependent responses at 239 nm for RP-UPLC-PDA separation of the oseltamivir phosphate/dexamethasone/daclatasvir dihydrochloride/remdesivir mixture.
e Peak retention time.f Peak capacity factor.g Peak tailing was evaluated at 5% (USP).h A drug peak's theoretical plate count.

Table S6 :
The preliminary stability study of the tested drugs in in different human plasma kits (K 3 EDTA, fluoride, and fluoride-EDTA)

st hour (Mean ± SD a , RSD %) 2 nd hour (Mean ± SD a , RSD %) 1 st hour (Mean ± SD a , RSD %) 2 nd hour (Mean ± SD a , RSD %) 1 st hour (Mean ± SD a , RSD %) 2 nd hour (Mean ± SD a , RSD %) 300
a Each result is the mean of 3 results in percentage ± their standard deviation in percentage.

Table S7 :
Application of the proposed method for the analysis of oseltamivir phosphate/dexamethasone/remdesivir mixture in human plasma (n = 6).The accuracy should be within ± 20% of the nominal concentration at the LLOQ and within ±15% at all the other levels.
a Conc.: Concentration.b The mean of six determinations.c a Each mixture contains equal concentrations of OSTP, DEX, and REM.

Table S9 :
The matrix effect (%ME), extraction efficiency (%RE), and process efficiency (%PE) and their IS normalized values for the tested drugs in fluoride-EDTA human plasma